A Methionine Chemical Shift Based Order Parameter Characterizing Global Protein Dynamics.

Coupling of side chain dynamics over long distances is an important component of allostery. Methionine side chains show the largest intrinsic flexibility among methyl-containing residues but the actual degree of conformational averaging depends on the proximity and mobility of neighboring residues. The 13 C NMR chemical shifts of the methyl groups of methionine residues located at long distances in the same protein show a similar scaling with respect to the values predicted from the static X-ray structure by quantum methods. This results in a good linear correlation between calculated and observed chemical shifts. The slope is protein dependent and ranges from zero for the highly flexible calmodulin to 0.7 for the much more rigid calcineurin catalytic domain. The linear correlation is indicative of a similar level of side-chain conformational averaging over long distances, and the slope of the correlation line can be interpreted as an order parameter of the global side-chain flexibility.

Determining methyl sidechain conformations in a CS-ROSETTA model using methyl 1H-13C residual dipolar couplings.

Modelling of protein structures based on backbone chemical shifts, using programs such as CS-ROSETTA, is becoming increasingly popular, especially for systems where few restraints are available or where homologous structures are already known. While the reliability of CS-ROSETTA calculations can be improved by incorporation of some additional backbone NMR data such as those afforded by residual dipolar couplings or minimal NOE data sets involving backbone amide protons, the sidechain conformations are largely modelled by statistical energy terms. Here, we present a simple method based on methyl residual dipolar couplings that can be used to determine the rotameric state of the threefold symmetry axis of methyl groups that occupy a single rotamer, determine rotameric distributions, and identify regions of high flexibility. The method is demonstrated for methyl side chains of a deletion variant of the human chaperone DNAJB6b.

NMR fragment screening reveals a novel small molecule binding site near the catalytic surface of the disulfide-dithiol oxidoreductase enzyme DsbA from Burkholderia pseudomallei.

The presence of suitable cavities or pockets on protein structures is a general criterion for a therapeutic target protein to be classified as 'druggable'. Many disease-related proteins that function solely through protein-protein interactions lack such pockets, making development of inhibitors by traditional small-molecule structure-based design methods much more challenging. The 22 kDa bacterial thiol oxidoreductase enzyme, DsbA, from the gram-negative bacterium Burkholderia pseudomallei (BpsDsbA) is an example of one such target. The crystal structure of oxidized BpsDsbA lacks well-defined surface pockets. BpsDsbA is required for the correct folding of numerous virulence factors in B. pseudomallei, and genetic deletion of dsbA significantly attenuates B. pseudomallei virulence in murine infection models. Therefore, BpsDsbA is potentially an attractive drug target. Herein we report the identification of a small molecule binding site adjacent to the catalytic site of oxidized BpsDsbA. 1HN CPMG relaxation dispersion NMR measurements suggest that the binding site is formed transiently through protein dynamics. Using fragment-based screening, we identified a small molecule that binds at this site with an estimated affinity of KD ~ 500 µM. This fragment inhibits BpsDsbA enzymatic activity in vitro. The binding mode of this molecule has been characterized by NMR data-driven docking using HADDOCK. These data provide a starting point towards the design of more potent small molecule inhibitors of BpsDsbA.

Optimized selection of slow-relaxing 13C transitions in methyl groups of proteins: application to relaxation dispersion.

Optimized selection of the slow-relaxing components of single-quantum 13C magnetization in 13CH3 methyl groups of proteins using acute (< 90°) angle 1H radio-frequency pulses, is described. The optimal selection scheme is more relaxation-tolerant and provides sensitivity gains in comparison to the experiment where the undesired (fast-relaxing) components of 13C magnetization are simply 'filtered-out' and only 90° 1H pulses are employed for magnetization transfer to and from 13C nuclei. When applied to methyl 13C single-quantum Carr-Purcell-Meiboom-Gill (CPMG) relaxation dispersion experiments for studies of chemical exchange, the selection of the slow-relaxing 13C transitions results in a significant decrease in intrinsic (exchange-free) transverse spin relaxation rates of all exchanging species. For exchanging systems involving high-molecular-weight species, the lower transverse relaxation rates translate into an increase in the information content of the resulting relaxation dispersion profiles.

Magic-Angle-Pulse Driven Separation of Degenerate 1 H Transitions in Methyl Groups of Proteins: Application to Studies of Methyl Axis Dynamics.

Dynamics of protein side chains is one of the principal determinants of conformational entropy in protein structures and molecular recognition events. We describe NMR experiments that rely on the use of magic-angle pulses for efficient isolation of degenerate 1 H transitions of the I=3/2 manifold of 13 CH3 methyl groups, and serve as 'building blocks' for the measurement of transverse spin relaxation rates of the fast- and slow-relaxing 1 H transitions - the primary quantitative reporters of methyl axis dynamics in selectively {13 CH3 }-methyl-labelled, highly deuterated proteins. The magic-angle-pulse driven experiments are technically simpler and, in the absence of relaxation, predicted to be 2.3-fold more sensitive than previously developed analogous schemes. Validation of the methodology on a sample of {13 CH3 }-labeled ubiquitin demonstrates quantitative agreement between order parameters of methyl three-fold symmetry axis obtained with magic-angle-pulse driven experiments and other established NMR techniques, paving the way for studies of methyl axis dynamics in human DNAJB6b chaperone, a protein that undergoes exchange with high-molecular-weight oligomeric species.

A Methyl-TROSY-Based 1 H Relaxation Dispersion Experiment for Studies of Conformational Exchange in High Molecular Weight Proteins.

Molecular complexes often sample conformational states that direct them to specific functions. These states can be difficult to observe through traditional biophysical approaches but they can be studied using a variety of different NMR spin relaxation experiments. However, these applications, when focused on moderate to high molecular weight proteins, are complicated by fast relaxing signals that negatively affect the sensitivity and resolution of spectra. Here a methyl 1 H CPMG-based experiment for studies of excited conformational states of protein machines is described that exploits a TROSY-effect to increase signal-to-noise. Complexities from the multiplicity of methyl 1 H transitions are addressed to generate a robust pulse scheme that is applied to a 320 kDa homeostasis protein, p97.

Determination of ligand binding modes in weak protein-ligand complexes using sparse NMR data.

We describe a general approach to determine the binding pose of small molecules in weakly bound protein-ligand complexes by deriving distance constraints between the ligand and methyl groups from all methyl-containing residues of the protein. We demonstrate that using a single sample, which can be prepared without the use of expensive precursors, it is possible to generate high-resolution data rapidly and obtain the resonance assignments of Ile, Leu, Val, Ala and Thr methyl groups using triple resonance scalar correlation data. The same sample may be used to obtain Met εCH3 assignments using NOESY-based methods, although the superior sensitivity of NOESY using [U-13C,15N]-labeled protein makes the use of this second sample more efficient. We describe a structural model for a weakly binding ligand bound to its target protein, DsbA, derived from intermolecular methyl-to-ligand nuclear Overhauser enhancements, and demonstrate that the ability to assign all methyl resonances in the spectrum is essential to derive an accurate model of the structure. Once the methyl assignments have been obtained, this approach provides a rapid means to generate structural models for weakly bound protein-ligand complexes. Such weak complexes are often found at the beginning of programs of fragment based drug design and can be challenging to characterize using X-ray crystallography.

Hyperpolarized nuclear Overhauser enhancement of alanine methyl groups by doubly relayed proton exchange.

Hyperpolarized water in dissolution dynamic nuclear polarization (dDNP) experiments has emerged as a promising method for enhancing nuclear magnetic resonance (NMR) signals, particularly in studies of proteins and peptides. Herein, we focus on the application of "proton exchange-doubly relayed" nuclear Overhauser effects (NOE) from hyperpolarized water to achieve positive signal enhancement of methyl groups in the side chain of an alanine-glycine peptide. In particular, we show a cascade hyperpolarization transfer. Initial proton exchange between solvent and amide introduces hyperpolarization into the peptide. Subsequently, intermolecular NOE relays the hyperpolarization first to Ala-Hα and then in a second step to the Ala-CH3 moiety. Both NOEs have negative signs. Hence, the twice-relayed NOE pathway leads to a positive signal enhancement of the methyl group with respect to the thermal equilibrium magnetization. This effect might indicate a way towards hyperpolarized water-based signal enhancement for methyl groups, which are often used for NMR studies of large proteins in solution.

An NMR Study of a 300-kDa AAA+ Unfoldase.

AAA+ ATPases are ubiquitous hexameric unfoldases acting in cellular protein quality control. In complex with proteases, they form protein degradation machinery (the proteasome) in both archaea and eukaryotes. Here, we use solution-state NMR spectroscopy to determine the symmetry properties of the archaeal PAN AAA+ unfoldase and gain insights into its functional mechanism. PAN consists of three folded domains: the coiled-coil (CC), OB and ATPase domains. We find that full-length PAN assembles into a hexamer with C2 symmetry, and that this symmetry extends over the CC, OB and ATPase domains. The NMR data, collected in the absence of substrate, are incompatible with the spiral staircase structure observed in electron-microscopy studies of archaeal PAN in the presence of substrate and in electron-microscopy studies of eukaryotic unfoldases both in the presence and in the absence of substrate. Based on the C2 symmetry revealed by NMR spectroscopy in solution, we propose that archaeal ATPases are flexible enzymes, which can adopt distinct conformations in different conditions. This study reaffirms the importance of studying dynamic systems in solution.

Precision measurements of relaxation rates of degenerate 1H transitions in methyl groups of small proteins.

An NMR experiment is designed for accurate and robust measurement of transverse relaxation rates of degenerate 1H transitions in selectively 13CH3-labeled, deuterated small proteins. The measurement is based on the use of acute (<90°) angle 1H radio-frequency pulses and relies on selection of the slow- and fast-relaxing components of methyl magnetization following the relaxation period in separate experiments. The R2 decay series recorded with selection of the fast-relaxing components serves as a useful complement to the R2 series acquired with selection of the slow-relaxing part, and permits the extension of the range of relative contributions of the fast- and slow-relaxing parts to apparent signal decay. The approach is experimentally verified on 13CH3 methyl groups of the ILV-{13CH3}-labeled protein ubiquitin at 10 °C and 25 °C. The obtained methyl 1H relaxation rates are in remarkably good agreement with the values obtained from well-established NMR techniques.

The measurement of relaxation rates of degenerate 1H transitions in methyl groups of proteins using acute angle radiofrequency pulses.

A simple NMR experiment for the measurement of transverse relaxation rates of degenerate 1H spins in 13CH3 methyl groups of deuterated proteins, is described. The experiment relies on the use of acute-angle 1H radio-frequency pulses, whereby a series of methyl 1H R2 decays is acquired with different values of the 1H pulse flip-angle, which modulates the relative contributions of the slow- and fast-relaxing components of 1H magnetization during the relaxation delay. These are subsequently analyzed simultaneously to extract the transverse relaxation rates of both components (RS and RF), along with the rate of cross-relaxation between these components, δ. The dipolar 1H-1H cross-correlated relaxation rate η = (RF - RS)/2 and the cross-relaxation rate δ, extracted from such acute-angle-pulse R2 decay series are compared with those derived from well-established methodology that uses relaxation-violated methyl 1H coherence transfer (so-called 'forbidden' experiments). Good agreement is achieved for the rates η derived from the two techniques, while slightly more accurate values of δ are obtained from analysis of acute-angle-pulse R2 series. Recording of acute-angle-pulse R2 series represents an attractive alternative to existing methodologies for quantifying methyl 1H relaxation and dynamics of the methyl three-fold symmetry axis in selectively [13CH3]-methyl-labeled, deuterated proteins - particularly so for very high-molecular-weight proteins, where the measurements of RF rates or the build-up of methyl 1H magnetization in relaxation-violated coherence transfer experiments, are problematic due to low sensitivity.

The WW1 Domain Enhances Autoinhibition in Smurf Ubiquitin Ligases.

Downregulation of ubiquitin (Ub) ligase activity prevents premature ubiquitination and is critical for cellular homeostasis. Nedd4 Ub ligases share a common domain architecture and yet are regulated in distinct ways through interactions of the catalytic HECT domain with the N-terminal C2 domain or the central WW domain region. Smurf1 and Smurf2 are two highly related Nedd4 ligases with ~70% overall sequence identity. Here, we show that the Smurf1 C2 domain interacts with the HECT domain and inhibits ligase activity in trans. However, in contrast to Smurf2, we find that full-length Smurf1 is a highly active Ub ligase, and we can attribute this striking difference in regulation to the lack of one WW domain (WW1) in Smurf1. Using NMR spectroscopy and biochemical assays, we identified the WW1 region as an additional inhibitory element in Smurf2 that cooperates with the C2 domain to enhance HECT domain binding and Smurf2 inhibition. Our work provides important insights into Smurf regulation and highlights that the activities of highly related proteins can be controlled in distinct ways.

The role of calcium in the interaction between calmodulin and a minimal functional construct of eukaryotic elongation factor 2 kinase.

Eukaryotic elongation factor 2 kinase (eEF-2K) regulates protein synthesis by phosphorylating eukaryotic elongation factor 2 (eEF-2), thereby reducing its affinity for the ribosome and suppressing global translational elongation rates. eEF-2K is regulated by calmodulin (CaM) through a mechanism that is distinct from that of other CaM-regulated kinases. We had previously identified a minimal construct of eEF-2K (TR) that is activated similarly to the wild-type enzyme by CaM in vitro and retains its ability to phosphorylate eEF-2 efficiently in cells. Here, we employ solution nuclear magnetic resonance techniques relying on Ile δ1-methyls of TR and Ile δ1- and Met ε-methyls of CaM, as probes of their mutual interaction and the influence of Ca2+ thereon. We find that in the absence of Ca2+ , CaM exclusively utilizes its C-terminal lobe (CaMC ) to engage the N-terminal CaM-binding domain (CBD) of TR in a high-affinity interaction. Avidity resulting from additional weak interactions of TR with the Ca2+ -loaded N-terminal lobe of CaM (CaMN ) at increased Ca2+ levels serves to enhance the affinity further. These latter interactions under Ca2+ saturation result in minimal perturbations in the spectra of TR in the context of its complex with CaM, suggesting that the latter is capable of driving TR to its final, presumably active conformation, in the Ca2+ -free state. Our data are consistent with a scenario in which Ca2+ enhances the affinity of the TR/CaM interactions, resulting in the increased effective concentration of the CaM-bound species without significantly modifying the conformation of TR within the final, active complex.

Allosteric fine-tuning of the conformational equilibrium poises the chaperone BiP for post-translational regulation.

BiP is the only Hsp70 chaperone in the endoplasmic reticulum (ER) and similar to other Hsp70s, its activity relies on nucleotide- and substrate-controllable docking and undocking of its nucleotide-binding domain (NBD) and substrate-binding domain (SBD). However, little is known of specific features of the BiP conformational landscape that tune BiP to its unique tasks and the ER environment. We present methyl NMR analysis of the BiP chaperone cycle that reveals surprising conformational heterogeneity of ATP-bound BiP that distinguishes BiP from its bacterial homologue DnaK. This unusual poise enables gradual post-translational regulation of the BiP chaperone cycle and its chaperone activity by subtle local perturbations at SBD allosteric 'hotspots'. In particular, BiP inactivation by AMPylation of its SBD does not disturb Hsp70 inter-domain allostery and preserves BiP structure. Instead it relies on a redistribution of the BiP conformational ensemble and stabilization the domain-docked conformation in presence of ADP and ATP.